Empirical and model-based evidence for a negligible role of cattle in peste des petits ruminants virus transmission and eradication

Peste des petits ruminants virus (PPRV) is a multi-host pathogen with sheep and goats as main hosts. To investigate the role of cattle in the epidemiology of PPR, we simulated conditions similar to East African zero-grazing husbandry practices in a series of trials with local Zebu cattle (Bos taurus indicus) co-housed with goats (Capra aegagrus hircus). Furthermore, we developed a mathematical model to assess the impact of PPRV-transmission from cattle to goats. Of the 32 cattle intranasally infected with the locally endemic lineage IV strain PPRV/Ethiopia/Habru/2014 none transmitted PPRV to 32 co-housed goats. However, these cattle or cattle co-housed with PPRV-infected goats seroconverted. The results confirm previous studies that cattle currently play a negligible role in PPRV-transmission and small ruminant vaccination is sufficient for eradication. However, the possible emergence of PPRV strains more virulent for cattle may impact eradication. Therefore, continued monitoring of PPRV circulation and evolution is recommended.

The isolate transmitted readily from sheep to sheep and from goat to goat (Trial 2).The co-housed animals all developed pyrexia and clear clinical signs with peak values for rectal temperature and clinical score at 10-12 and 7-11 days, respectively, after the corresponding peak for the inoculated animals.Similarly, seroconversion was detected by 7 dpi for inoculated animals and by 17 dpi for sentinel animals (Figure S5).The lag observed for measures of virus excretion (qRT-PCR) was shorter (5-9 days for sheep, 1-6 days for goats; NB: LOESS smooth did not capture first goat peak in whole blood well so modified range for all goat measures is likely 4-7 days).PPRV was isolated in cell culture from swabs collected on 4, 7, 10 dpi from inoculated animals in trials 1 or 2 (Figure S4 and S5, red crosses) and on 10, 14, and 17 dpi from co-housed animals (trial 2, Figure S5, red circles).All the co-housed animals were infected with PPRV and developed the same level of disease seen in the inoculated animals.When comparing antigen ELISA and qRT-PCR results from nasal swabs taken from both inoculated and co-housed sheep and goats, findings were comparable in trajectory, dpi of peak value, inter-peak interval in days, and uncertainty (Figure S8).

Challenge experiment and controls confirm expected disease progression and facility biosecurity
Seropositive and seronegative goats remaining from trial 3 were challenged with PPRV 13 days after the end of trial 3. The 3 PPRV-seropositive goats were placed in the same barn and were all protected from disease upon challenge.The 7 seronegative goats were not moved and developed clinical signs of PPR and seroconverted upon challenge (Figure S9).No virus isolation data were collected during the challenge experiment and no animals died.
Positive (inoculated) and negative (PPRV seronegative) control animals (see Methods) from each trial were run at the same time in a randomly assigned barn.The results from these control animals in trials 1-2 (Figures S4-S5) and trials [3][4][5] were as expected with the positive controls developing PPR (clinical signs, seroconversion, RNA shedding) and negative controls showing no detectable clinical signs of PPR or seroconversion.

Virus
The PPRV lineage IV (LIV) isolate was stored as a pooled sample of nasal and ocular swabs and gum debris according to Alemu et al.

Animals and Study Design
Markets animals were purchased from were in Western and Northern Showa and included Muger (Inchiny), Ginchi-Dendi, Ejere, and Degem.Sellers reported bringing animals to market from within a 20 km radius Animals were housed on the AHI campus and baseline serological testing for PPRV was conducted to confirm seronegative status.Water and hay were provided ad libitum and refreshed daily.On each day, animals were fed, the barn cleaned of old feed and waste, the rectal temperatures of all animals was determined, and animals were monitored for clinical signs.
All animals were infected intranasally using a 3mL syringe fitted with a nasal atomizer (MAD-300, Dixie EMS Supply).Nasal, ocular, and rectal swabs were collected in duplicate and sent directly to the molecular and cell culture laboratories for processing.Swabs were collected using dry cotton or synthetic swabs and directly soaked in 1 ml of DMEM with 2% fetal bovine serum (FBS), and 1x (ocular and nasal swab) or 5x (rectal swab) antibiotic and antimycotic (Corning: 10,000 units/ml penicillin, 10mg/ml streptomycin sulfate, 25 μg/ml amphotericin B combination, VWR 45000-616).
Negative controls were used in each trial to have comparative rectal temperature, clinical signs, and serology data and assess biosecurity practices to ensure PPRV was not spreading between barns.Positive controls were used to check for expected transmission between small ruminants or expected clinical signs from isolate passage used.To reduce the number of animals used, the number of controls was reduced over the course of the five trials.For Trials 1-2 there were 3 each of sheep and goat negative controls.For Trial 3 there were positive controls (4 inoculated, 4 sentinel goats), and 3 each of cattle and goat negative controls.For Trial 4 there were positive controls (2 inoculated, 2 sentinel goats), and 2 goat negative controls.For Trial 5 there were 2 inoculated goat positive controls and 2 goat negative controls.

Serological and Molecular Analysis
Serological analysis was conducted using the ID Screen PPR Competition kit (IDvet PPRC-4P and PPRC-10P, France).
For molecular analysis, RNA was extracted using the QIAamp Viral RNA Mini kit (Qiagen 52904 and 52906), and real time quantitative RT-PCR conducted with custom Taqman QSY probe (Thermofisher 4482777, FAM-5-CACCGGAYACKGCAGCTGACTCAGAA-3-QSY) and Express One Step kit (Thermofisher 1178101K) on an Applied Biosystems 7500 FAST instrument.The forward and reverse primers of the PPRV N gene were 5-AGAGTTCAATATGTTRTTAGCCTCCAT-3 and 5-TTCCCCARTCACTCTYCTTTGT-3, respectively.To observe molecular dynamics during the trials, swabs were tested by sandwich ELISA (ID Screen PPR Antigen Capture, IDvet PPRAG-2P, France).Antigen testing with this kit could be run more rapidly after sampling than qRT-PCR testing, which was typically conducted after a trial was complete.Antigen

Text S3: Experimental Settings Facility
The experimental facility has six experimental barns inside the facility, three on each side of a central hallway (Figure S3).Each barn had an enclosed, small courtyard (approximately 4 meters by 3 meters) with no roof and a closed, roofed barn (approximately 8 meters long, 3 meters wide) with skylight and wire mesh ventilation that allowed air to enter the front or back, but not between barns.During acclimatization the animals were allowed to roam in their assigned barn courtyard (no roof) and inside their assigned roofed barn; however, upon the start of the trial, animals were solely kept inside the roofed barns.The maximum number of animals in a barn was 8 (4 inoculated, 4 co-housed PPRV seronegative animals).Water and feed were provided ad libitum and refreshed daily and the barn was cleaned of debris and fecal material.Specifically, a half bail of hay (6kg) and 2kg concentrate per calf per day and 3kg hay and 1 kg concentrate per sheep/goat per day.From the second trial onward, the barn-length feeding trough had metal bars to discourage sheep and goats from jumping into feeding trough.In cold weather, grass was added for bedding and drafts around the door were blocked with additional boards.

Biosecurity
All personnel entering the facility, including animal attendants, were required to shower to enter and exit the facility (Figure S3A).All personnel wore boots, scrubs, a full body Flexothane suit (Sioen Montreal coverall 4964A2FC1), and N95 masks (masks starting in Trial 2, March 2020) for use solely on this project while in the facility.A footbath was present to be used upon entry and exit just after the showers and at the entrance to the central hallway.For each barn (Figure S3B) there were two footbaths, one just inside the door of the courtyard from the central hallway and one just in front of the door to the roofed barn and all personnel stepped in both baths on the way in or out of the barns.Each animal attendant was assigned to a specific barn and did not enter other barns when a trial was actively running, whereas the sampling team of veterinarians and researchers (typically 3 or less) moved between barns for sampling, which started in the morning and ended by early afternoon.In total, to enter or exit a particular barn, personnel stepped in 4 footbaths filled with a dilution of FAM-30 disinfectant (Evans Vanodine, R067 KEV).When exiting any barn, while stepping in the first footbath, the sampling team and barn-specific attendants assisted each other to spray and sponge down their Flexothane suits with FAM-30 dilutions available in spray bottles.They waited for the suits to dry before entering the courtyard of the next barn.Clinical signs data was recorded by photo of a whiteboard in each barn and of each animal (photos included: eartag, head profile, frontal profile, conjunctival tissue, oral view, rectal view).A Go-Pro Hero 7 camera in waterproof housing case, used to record photos and video of clinical data and signs in the barn, was also sprayed with FAM-30 disinfectant on the way out of each barn, as were any items leaving the barn with the sampling team.Each barn had its own supply of latex gloves, biohazard trash bags, trash container, whiteboard makers and erasers, sample labelling markers, and broom that stayed within the barns.New gloves were put on by the personnel once they arrived in each barn and these gloves were discarded before leaving a barn.Prepared racks with labeled blood and serum vials and cryovials for collecting molecular swabs where only brought into the assigned barns, sprayed with FAM-30 upon exit, and stored in a cooler with icepacks while sampling was completed.They were then transferred immediately to AHI sample reception and appropriate laboratories.At the end of a trial, barns were cleaned of feed and waste and were sprayed down with FAM-30 disinfectant and left to dry before moving on to the next trial.Each barn had its own drain in the floor to take liquid waste to a tank on the side.

Text S4. Power analysis details to determine detectable probability of cattle-to-goat transmission
Transmission is a binary event that can be modeled by binomial distribution Bin(n, p) where the distribution is the number of successes (transmissions) in n number of independent trials with a probability p of success (transmission) for each trial.In this trial there were 32 cattle goat pairs (trials).We simulated 1000 trial outcome observations, summing the outcomes and divided by 1000 to obtain an average, simulated a range of probabilities of cattle-to-goat transmission (we used 0.0001 to 0.1 or 0.01-10%), and plotted these values.We drew a horizontal line on the y axis at 0.8 -indicating the cutoff where there was an 80% chance of detecting cattle-to-goat transmission, if it occurred.The vertical line intersects the simulated data at a probably of cattleto-goat transmission of 5%, which is the lowest probability of transmission this study is powered to detect.# creates an empty vector of length 100 filled with NA. this vector will hold the y axis values, the probability of detecting transmission.
q <-rep(NA,100); # creates a vector of length 100 filled with a sequence of numbers to represent low probability values which ranged between 0.0001 and 0.1.this vector will hold the x-axis values, the probability of C->G transmission.
pp <-seq(0.0001,0.1,length= 100); # loops over the 100 indexed spots in the q vector and generates random draws from a binomial distribution (1000 observations of 32 trials with probabilities greater than 0 given in the pp vector), which are summed and divided by 1000 to generate an average value, and the average value is placed into the respective spot in the empty q vector) for(i in 1:100){q[i] <-sum(rbinom(1000,32,pp[i])>0)/1000} # plots pp and q vector and adds text for x and y axes plot(pp,q, xlab = "Probability of C -> G Transmission", ylab= "Probability of Detecting") # adds vertical and horizontal lines to the plot abline(v=0.05)abline(h=0.8)

Fig. S1. Power analysis to determine detectable probability of cattle-to-goat transmission.
One thousand simulated draws for a range of cattle (C) to goat (G) transmission probabilities (xaxis) were simulated from a binomial distribution with trial size 32.The horizontal line on the yaxis represents a 80% chance of detecting transmission, if it exists.The vertical line intersects the simulated data and horizontal line at a probably of cattle-to-goat transmission of 5%, which is the lowest probability of cattle-to-goat transmission this study was powered to detect.Sampling days on which PPRV could be isolated (cross if isolated from inoculated animal, circle for sentinel animal) are indicated with red and dpi with deaths are indicated with black (cross for inoculated animal deaths, circle for sentinel deaths, x for euthanized) along the x-axis.Isolation was not conducted on whole blood samples.One inoculated goat was euthanized.Vertical lines indicate day post infection (dpi) of peak value and inter-peak interval (difference in dpi of peak value for each animal group) is indicated in black.Sampling days on which PPRV could be isolated (cross if isolated from inoculated animal, circle for sentinel animal) are indicated with red and dpi with deaths are indicated with black (cross for inoculated animal deaths, circle for sentinel deaths, x for euthanized) along the x-axis.Isolation was not conducted on whole blood samples.One death occurred in an inoculated goat.explores a symmetric transmission rate within each species (βSS = βSC = 1.9 x 10 -1 ; βCC = 2.1 x 10 -3 ; βCS varies 0-0.19).).Scenario 3 (C) explores asymmetric transmission rate between species (βSS = 1.9 x 10 -1 ; βSC = βCC =2.1 x 10 -3 ; βCS varies 0-0.19).).Lastly, scenario 4 (D) explores asymmetric transmission rate within and between species (βSS = 1.9 x 10 -1 ; βSC = 9.9 x 10 -2 ; βCC = 0; βCS varies 0-0.19).).See Methods, Text S5, Text S6, Table S2 for more information on parameter values selected and model code results from Trial 2 (Fig S8), the post Trial 3 challenge (Fig S9), all cattle-to-goat transmission trial barns (Fig S13), and positive control barns in Trials 3-5 (Figs S10-12) were visualized.

Fig. S2 .
Fig. S2.Study design of Trials 1-5.The series of trials tested that PPRV/Ethiopia/Habru/2014 could infect, cause clinical signs and mortality, and transmit among small ruminants before addressing the open question of cattle spillback transmission to goats directly in Trials 3-5.Controls are not shown but were randomly assigned to one of the six barns in the facility and monitored concurrently with experimental barns during each trial.Darker colored animals with syringes are inoculated, lighter colored animals without syringes are co-housed, PPRVseronegative animals.

Fig. S5 .
Fig. S5.Within host and transmission dynamics of PPRV among co-housed local breeds of Ethiopian sheep and goats.Trial 2 A. daily rectal temperature, B. daily clinical score, C.serology (competitive ELISA), and D. viral RNA from ocular, nasal, and rectal swabs and whole blood (RT-PCR).Thin lines represent individual animals and bold lines represent smooth local regression (LOESS) curves of all animals in the category (control, inoculated, sentinel).Gray shading indicates 95% confidence bands (t-based approximation).Vertical lines indicate day post infection (dpi) of peak value and inter-peak interval (difference in dpi of peak value for each animal group) is indicated in black.Sampling days on which PPRV could be isolated (cross if isolated from inoculated animal, circle for sentinel animal) are indicated with red and dpi with deaths are indicated with black (cross for inoculated animal deaths, circle for sentinel deaths, x for euthanized) along the x-axis.Isolation was not conducted on whole blood samples.One death occurred in an inoculated goat.

Fig. S6 .Figure S7 .
Fig. S6.Impact of rectal temperature (A) on clinical score calculated with (B) and without (C) rectal temperature shows that the rise and peak of clinical score does not differ for sheep and goats, though the return to baseline clinical score may take longer for sheep (C).Combined sheep and goat data from Trials 1 and 2. Thin lines represent individual animals and bold lines represent smooth local regression (LOESS) curves of all animals in the category (inoculated, sentinel).Gray shading indicates 95% confidence bands (t-based approximation).Vertical lines indicate day post infection (dpi) of peak value and inter-peak interval (difference in dpi of peak value for each animal group) is indicated in black.

Fig. S8 .
Fig. S8.Comparison of RNA detection between antigen ELISA (A) RT-PCR (B) among nasal swabs from inoculated and sentinel sheep and goats from Trial 2. Thin lines represent individual animals and bold lines represent smooth local regression (LOESS) curves of all animals in the category (inoculated, sentinel).Gray shading indicates 95% confidence bands (tbased approximation).Vertical lines indicate day post infection (dpi) of peak value and inter-peak interval (difference in dpi of peak value for each animal group) is indicated in black.

Fig. S10 .
Fig. S10.Trial 3 clinical, serological, and molecular results from positive control goats (A) and clinical and serological data from negative control cattle and goats (B).From left to right in each row there is measurements of daily rectal temperature, daily modified clinical score, serology (competitive ELISA), and viral RNA from ocular, nasal, and rectal swabs measured on antigen ELISA.No antigen ELISA were run on negative control animals as serology showed they were all negative.Thin lines represent individual animals and bold lines represent smooth local regression (LOESS) curves of all animals in the category (inoculated, sentinel).Gray shading indicates 95% confidence bands (t-based approximation).When present, vertical lines indicate day post infection (dpi) of peak value and inter-peak interval (difference in dpi of peak value for each animal group) is indicated in black.Sampling days on which PPRV could be isolated (cross if isolated from inoculated animal, circle for sentinel animal) are indicated with red and dpi with deaths are indicated with black (cross for inoculated animal deaths, circle for sentinel deaths, x for euthanized) along the x-axis.Five positive controls died in total (3 inoculated [2 on dpi 10], 2 sentinel); one of the two sentinels was euthanized (dpi 17).

Fig. S11 .
Fig. S11.Trial 4 clinical, serological, and molecular results from positive control goats (A) and clinical and serological data from negative control cattle and goats (B).From left to right in each row there is measurements of daily rectal temperature, daily modified clinical score, serology (competitive ELISA), and viral RNA from ocular, nasal, and rectal swabs measured on antigen ELISA.No antigen ELISA were run on negative control animals as serology showed they were all negative.Thin lines represent individual animals and bold lines represent smooth local regression (LOESS) curves of all animals in the category (inoculated, sentinel).Gray shading indicates 95% confidence bands (t-based approximation).Sampling days on which PPRV could be isolated (cross if isolated from inoculated animal, circle for sentinel animal) are indicated with red and dpi with deaths are indicated with black (cross for inoculated animal deaths, circle for sentinel deaths, x for euthanized) along the x-axis.Four positive controls died in total (2 inoculated, 2 sentinel [dpi18 and dpi 20]).

Fig. S12 .
Fig. S12.Trial 5 clinical, serological, and molecular results from positive control goats (A) and clinical and serological data from negative control cattle and goats (B).From left to right in each row there is measurements of daily rectal temperature, daily modified clinical score, serology (competitive ELISA), and viral RNA from ocular, nasal, and rectal swabs measured on antigen ELISA.No antigen ELISA were run on negative control animals as serology showed they were all negative.Thin lines represent individual animals and bold lines represent smooth local regression (LOESS) curves of all animals in the category (inoculated, sentinel).Gray shading indicates 95% confidence bands (t-based approximation).When present, vertical lines indicate day post infection (dpi) of peak value and inter-peak interval (difference in dpi of peak value for each animal group) is indicated in black.Sampling days on which PPRV could be isolated (cross if isolated from inoculated animal, circle for sentinel animal) are indicated with red and dpi with deaths are indicated with black (cross for inoculated animal deaths, circle for sentinel deaths, x for euthanized) along the x-axis.One inoculated positive control died.

Fig. S13 .
Fig. S13.Trials 3 to 5 antigen ELISA results from nasal (A) and rectal swabs (B) from inoculated cattle and sentinel goats.Thin lines represent individual animals and bold lines represent smooth local regression (LOESS) curves of all animals in the category (inoculated, sentinel).Gray shading indicates 95% confidence bands (t-based approximation).

Fig. S14 .Figure S16 .
Fig. S14.Posterior distributions of the transmission probabilities observed in the empirical trials, calculated from a beta binomial Bayesian model using bayesrules R package (see Methods, Text S5).Posterior distributions are in green, uniform uninformative priors are in yellow, and the scaled likelihood is in blue (and completely overlapped by the posterior).Cattle (C), small ruminants (S).

Table S1 .
Concentrations and passage information for isolations used in all experiments.